Method for synthesis of oligosaccharides

ABSTRACT

A method for synthesis of oligosaccharide compounds which either consist of or are fragments or analogs of the carbohydrate part in glycoconjugates is described. The synthesis from donor and acceptor substrates is carried out in that at least one glycosidase and at least one glycosyltransferase are used as catalysts and the oligosaccharide compound is isolated from the reaction mixture.

DESCRIPTION

The present invention relates to a method for enzymatic synthesis of anoligosaccharide compound, which either consists of or is a fragment oran analog of the carbohydrate part in a glycoconjugate. Furthermore, theinvention relates to the use of the product prepared by this method.

It has been found that the oligosaccharide part of variousglycoconjugates (especially glycolipids and glycoproteins) have a numberof important functions in vivo (Biology of Carbohydrates, Vol. 2.,Ginsburg et al., Wiley, New York (1984); The Glycoconjugates, Vol. 1-V,Academic Press, New York; S. Hakomori, Ann. Rev. Biochem. Vol. 50, pp.733-64); Feizi, Nature, pp. 314 (1985); S. Hakomori, Chemistry andPhysics of Lipids, Vol. 42, pp. 209-33). Among other things it was foundthat

the carbohydrate structures are important for the stability, activity,localisation, immunogenicity and degradation of glycoproteins;

carbohydrates are antigenic determinants (for example blood groupantigens);

carbohydrates function as receptors when bound to cell surfaces forpathogens, proteins, hormones, toxins and during cell-cell interactions;

carbohydrates are important to oncogenesis, since specificoligosaccharides have been found to be cancer-associated antigenicdeterminants;

frequently only a smaller sequence (di- or trisaccharide) of thecarbohydrate part of the glycoconjugate is required for full biologicalactivity (e.g. receptor activity).

Universities and industry are at present working intensely on developingthe use of biologically active oligosaccharides within a number ofdifferent fields, such as

novel diagnostics and blood typing reagents

highly specific materials for affinity chromatography

cell specific agglutination reagents

targeting of drugs

monoclonal antibodies, specific against e.g. cancer-associatedstructures

therapy

Besides the above-mentioned fields, a considerable future market isenvisaged for fine chemicals based on biologically active carbohydrates.

The organic chemical techniques used today for synthesis of thesecarbohydrate structures require an extensive protective group chemistrywith many steps of synthesis and expensive catalysts. Low total yieldsare obtained in those complicated reaction schemes and the technique isnot favorable, especially for larger scale work.

Enzymes are nature's own catalysts with many attractive characteristics,such as high stereo-, regio- and substrate selectivity as well as highcatalytic efficiency under mild conditions. Today, great hopes aretherefore placed in being able to utilize enzymes for large-scaleselective synthesis of oligosaccharides with fewer reaction steps andconsequently higher total yields than by organic chemical methodology.

Both hydrolases (glycosidases. EC 3.2) and glycosyltransferases (EC 2.4)can be used for synthesis (glycosidases: see Nisizawa et al. in TheCarbohydrates, Chemistry and Biochemistry, 2nd Ed., Vol. IIA, pp.242-290, Academic Press, New York (1970)). With glycosidases reversedhydrolysis (equilibrium reaction) or transglycosylation (kineticreaction) are often used to obtain synthesis (see e.g. K.G.I. Nilsson,Carbohydrate Res., Vol. 167, pp. 95-103 (1987)). With transferases anucleotide sugar (UDP-Gal, CMP-Sia, UDP-GalNAc, GDP-Fuc, etc), which isrelatively expensive, is used as donor. Both types of enzymes haveadvantages. Glycosidases are abundant and can often be used directlywithout purification, glycosyltransferases show high regio- andacceptor-selectivity. However, both types of enzymes have disadvantageswhen used for synthesis. Glycosidases have a low or often wrongregioselectivity which may result in complicated product mixtures andthus purification problems. As a result glycosidases are often notsuitable for synthesis of higher oligosaccharides. Glycosyltransferasesare often present in small amounts in living cells and are thus often oflow availability. Furthermore, as mentioned above, the transferases arecofactor dependent.

One of the objects of the present invention is to use the properties ofglycosidases and glycosyltransferases in a favorable way for efficientsynthesis of oligosaccharides. This is achieved according to theinvention by combining glycosidase-catalysed synthesis of anoligosaccharide compound with glycosyltransferase-catalysed synthesis ofthe final, higher oligosaccharide. An easily available glycosidase isthus used for synthesis of the shorter oligosaccharide compound and aregiospecific enzyme (i.e. glycosyltransferase) is used when a higherregioselectivity is required, i.e., for synthesis of the finaloligosaccharide. This is illustrated in the following scheme (which isnot intended to restrict the scope of the invention); ##STR1## (D₁ R₁symbolizes donor saccharide (oligosaccharide, glycoside) with α- orβ-bound aglycon (R₁), D₁ AR₂ is an O--, C--, N--, S-- or F-glycoside ofa di- or higher oligosaccharide, ND₂ is a suitable sugar nucleotide(CMP-Neu5Ac, UDP-Gal, UDP-GalNAc, GDP-Fuc, etc) and D₂ D₁ AR₂ is thefinal oligosaccharide product. The glycosidase reaction can also be anequilibrium reaction. More than one glycosidase and/or transferase canbe used for synthesis of higher oligosaccharides.

The substrates are selected with regard to the oligosaccharide which isto be synthesized, and are often commercially available or can besynthesized by organic or enzymatic synthesis and therefore do notrestrict the use of the invention.

The enzymes are selected with regard to the final oligosaccharide whichis to be synthesized. The enzyme can be used in situ (especially severalglycosidases) or after partial or complete purification (especiallyglycosyltransferases) from their natural environment. The enzyme may beused in soluble form or immobilized to a solid phase by e.g. adsorption,encapsulation, chelation, precipitation or covalent binding.Simultaneous use of glycosidase and glycosyltransferase in soluble formor immobilized to a solid phase (eventually co-immobilized) may beadvantageous according to the invention facilitating the conversion ofthe intermediate oligosaccharide product (e.g. D₁ AR₂ in the schemeabove) to the final product oligosaccharide (e.g. D₂ D₁ AR₂ in thescheme above). In this way the method according to the invention givesimportant advantages compared to previous methods: purification ofintermediary product is not necessary, secondary hydrolysis is minimized(i.e. higher yield) and trisaccharides or higher oligosaccharides can besynthesized in a minimum of "pots" (in some cases one-pot reactions).This is facilitated by the high acceptor specificity of mostglycosyltransferases: the transferase in the scheme above does not reactwith the wrong isomer of D₁ AR₂ or with D₁ R₁. Thus, for example,CMP-N-acetylneuraminate-β-D-galactoside (α2-3)sialyltransferase (EC2.4.99.4) prefers Galβ1-3GalNAcR over Galβ1-3GlcNAcR as acceptor andGalβ1-4GlcNacR and GalR are poor acceptors (Sadler et al., J. Biol.Chem., Vol. 254, pp. 4434-43). If glycosides (AR₂) are used as acceptorsin the glycosidase-catalysed reaction, a product glycoside is obtainedwhich is easy to purify since no anomerisation of the product glycosideoccurs. Furthermore, the same glycosidase may be used for predominantsynthesis of several isomers, since the regioselectivity may be changedby the use of different aglycons and by changing the configuration (α-or β-) of the glycosidic linkage between for example A and R₂ in thescheme above (K.G.I. Nilsson, Carbohydrate Res., Vol. 167, pp. 95-103).The aglycon R₂ may be an organic compound of varying type (aliphatic,aromatic, heterocyclic, or variations thereof) which is O--, N--, C--,S-glycosidically bonded to A. R₂ may also be glycosidically bound F oran --OH group.

As examples of suitable organic aglycons mention may be made of CH₃(CH₂)n-groups (methyl, ethyl, etc.), 2-bromoethyl, allyl,trimethylsilylethyl, 2-(2-carbometoxiethylthio)ethyl, amino acids(seryl, threonyl, asparaginyl, etc) or derivatives thereof, peptides,phenyl, bensyl, nitroophenyl, lipids and analogs thereof.

Examples of α- and β-glycosidases which may be used according to theinvention are D-mannosidases, D-galactosidases, L-fucosidases,N-acetyl-D-galactosaminidases, hexosaminidases and other glycosidases inthe EC group 3.2 (Enzyme Nomenclature, Academic Press, 1984).

Examples of sialyl-, galactosyl-, fucosyl-, N-acetyl-glucosaminly-,N-acetyl-galactosaminyl- and mannosyltransferases which can be usedaccording to the invention are found in the EC group 2.4 (EnzymeNomenclature, Academic Press, 1984). Recombinant enzymes can be usedaccording to the invention.

The synthesis method according to invention is generally applicable tothe synthesis of oligosaccharide sequences included in glycoconjugates(see examples of structures given in references on page 1 one above). Ofspecial interest are the minutest fragments of these structures, whichare sufficient to transfer biological activity and the choice of Di andA in the scheme above is determined by this.

Examples of interesting structures are blood group determinants,cancer-associated oligosaccharide structures and structures withbiological receptor activity (see references on p. 1).

Some examples of how the invention may be used in actual practice aredescribed in the following Examples, which however are in no wayintended to restrict the scope of the invention (abbreviations accordingto IUPAC-IUB's recommendations, J. Biol. Chem., Vol. 257, pp. 3347-3354(1982)).

EXAMPLE 1

Synthesis of Neu5Ac(α2-3)Gal(β1-3)GalNAc(β)-OEtBr. GalNAc(β)-OEtBr wasobtained by mixing GalNAc(β)-OPhNO₂ -p (1.2 g) in 100 ml sodiumphosphate buffer (0.05 M, pH 5.2) with 2-bromoethanol (10 ml) and addingN-acetyl-β-D-glucosaminidase (EC 3.2.1.30; 70 U).

After 48 h at room temperature 500 mg of GalNAc(β)-OEtBr was isolated bycolumn chromatography (Kieselgel 60, Merck; methylenechloride-methanol-water). GalNAc(β)-OEtBr (400 mg) and Gal(β)-OPhNO₂ -o(1 g) were suspended in 13 ml 0.03 M sodium phosphate buffer, pH 6.5,and dimethylformamide (4 ml) and 7.2 ml β-D-galactosidase from bovinetestes (2 U; Sigma) were added. After 4 days at 37° C. the product wasisolated by column chromatogrphy as above. The fractions containingproduct was acetylated and further purified by column chromatography.Deacetylation gave 40 mg pure Gal(β1-3)GalNAc(β)-OEtBr which wascharacterized with NMR (¹³ C, ¹ H).

CMP-Neu5Ac (4 mg, enzymatically prepared) and the above disaccharide (4mg) were dissolved in 2 ml 0.1 M MES-HCl, pH6.7,CMP-N-acetylneuraminyl-β-D-galactoside (α2-3)sialyltransferase (EC2.4.99.4, porcine submaxillary gland, 20 mU. 0.17 ml, Genzyme) was addedtogether with 10 μl Triton X-100 and 2 mg bovine serum albumin. MoreCMP-Neu5Ac (4 mg) was added after 20 h. After a total reaction time of72 h at 37° C. 5 mg of Neu5Ac(α2-3)Gal(β1-3)GalNAc(β)-OEtBr was isolatedby column chromatography (Kieselgel 60, acetonitrile-2-propanol-2.5 MNH₄ OH and Sephadex G-15). This product was pure according to NMR (¹ H,¹³ C) and the structure was confirmed by methylation analysis.

EXAMPLE 2

Synthesis of Neu5Ac(α2-3)Gal(β1-3)GlcNAc(β)-OMe.

This substance was prepared analogously. β-D-galactoside (α2-3)sialyltransferase (0.34 ml, 40 mU) was added to 0.7 ml 0.1 M MES-CHl, pH6.7, which contained 30 mg Gal(β1-3)GlcNAc(β)-OMe (synthesized asdescribed above for Gal(β1-3)GalNAc(β)-OEtBr but with GlcNAc(β)-OMe asacceptor), 10 mg CMP-Neu5Ac, 5 μl Triton X-100 and 1 mg albumin. MoreCMP-Neu5Ac (10 mg) was added after 30 h. After five days at 37° C.column chromatography as described above gave 10 mgNeu5Ac(α2-3)Gal(β1-3)GlcNAc(β)-OMe which was pure according to NMR. Thestructure was confirmed with NMR and methylation analysis.

The acceptor selectivitity of the glycosyltransferases in several of theexamples is such that co-immobilized glycosidase and glycosyltransferasecan be used and in some cases one-pot reactions are possible (enzymes,glycosidase substrate and nucleotide sugar which are used are mixeddirectly or nucleotide sugar and glycosyltransferase are added after theglycosidase). In some cases the glycosidase product is only partiallypurified (i.e. Sephadex G10 column) before further reaction with theglycosyltransferase.

EXAMPLE 3 Synthesis of Neu5Acα2-3Galβ1-4GlcNAcβ-OMe

β-D-Galactosidase from bovine testes was used as in Example 2 forsynthesis of Galβ1-4GlcNAc-OMe (the enzyme gives this isomer in additionto the β1-3 isomer; β-galactosidas from another source (lactobacillus orsporobolomyces) is used for more specific synthesis ofGalβ1-4GlcNAc-OMe).

α2-3Sialyltransferase (EC 2.4.99.5) is used for the sialylation.

EXAMPLE 4 Synthesis of Neu5Acα2-6Galβ1-4GlcNAcβ-OMe

Synthesis as in example 3 but with α2-6sialyltransferase (EC 2.4.99.1).

EXAMPLE 5 Synthesis of Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα-OEtBr

Synthesis as in example 1 using GalNAcα-OEtBr as acceptor and inaddition α2-6sialyltransferase (EC 2.4.99.7) as catalyst.

EXAMPLE 6 Synthesis of Fucα1-2Galβ1-3GlcNAcβ-OMe

Synthesis as in example 2 but with α1-2fucosyltransferase (EC 2.4.1.69)and GDP-Fuc as donor instead of α2-3sialyltransferase and CMP-Neu5Ac,respectively.

EXAMPLE 7 Synthesis of Fucα1-2Galβ1-4GlcNAcβ-OMe

Synthesis as in example 6 but with β1-4 isomer as acceptor.

EXAMPLE 8 Synthesis of Galβ1-4(Fucα1-3)GlcNacβ-OMe

Synthesis as in example 7 but with α2-3fucosyltransferase (EC 2.4.1.152or EC 2.4.1.65 with which also Galβ1-3(Fucα1-4)GlcNAc can be synthesizedas in example 6) as catalyst.

EXAMPLE 9 Synthesis of Galα1-3Galβ1-4GlcNAcβ-OMe

Synthesis as in example 3 but instead of EC 2.4.99.6 and CMP-Neu5Ac,α1-3galactosyltransferase (EC 2.4.1.151) and UDP-Gal are used.

EXAMPLE 10 and 11 ##STR2##

Synthesis as in example 6 (substance 10; blood group A, type 1) and asin example 7 (substance 11; blood group A, type 2), respectively, and inaddition to EC 2.4.1.69, α1-3N-acetylgalactosaminyltransferase (EC2.4.1.40, from e.g. human milk) and UDP-GalNAc are used.

EXAMPLE 12 and 13 ##STR3##

Substance 12 and 13 (blood group B, type 1 and 2, respectively) aresynthesized as 10 and 11, respectively, but with EC 2.4.1.37 and UDP-Galinstead of EC 2.4.4.1.40 and UDP-GalNac.

In the above examples various methyl and bromoethyl glycosides wereprepared. The expert can easily synthesize other interesting glycosidesexemplified in the description and choose optimal conditions for thereactions. The sugar nucleotides are obtained with e.g. enzymaticsynthesis (nucleotidyltransferase+nucleotide +monosaccharid (ormonosaccharide-1-phosphate) for example CTP+Neu5Ac+CMP-Neu5Ac-syntas (EC2.7.7.43).

I claim:
 1. A method of synthesizing an oligosacchride compound productwhich either consists of or is a fragment or analog of the carbohydratepart in a glycoconjugate, said method consisting essentially ofreacting(a) at least one donor substance comprising a oligosaccharide ormonosaccharide or glycoside, (b) at least one acceptor substancecomprising a monosaccharide, oligosaccharide, glycoside, or saccharideanalog, (c) at least one nucleotide sugar which is a donor substance,and (d) at least one E.C. group 3.2 glycosidase and at least one E.C.group 2.4 glycosyltransferase, to synthesize said oligosaccharidecompound product, whereby said glycosidase enters into atransglycosylation or reversed hydrolysis reaction to thereby synthesizesaid oligosaccharide compound product.
 2. The method as claimed in claim1, wherein the carbohydrate portion of said donor substance in (a) or(c) and said acceptor substance in (b) is at least one member selectedform the group consisting of D-galactose, D-mannose, N-acetylneuraminicacid, N-acetyl-D-galactosamine, N -acetyl-D-glucosamine, L-fucose, andanalogs thereof.
 3. The method as claimed in claim 1, wherein saidacceptor substance is a glycoside in which the aglycon is glycosidicallybound fluorine or an O--, N--, C-- or S-- glycosidically bound aliphaticor aromatic compound.
 4. The method as claimed in claim 3, wherein saidaliphatic or aromatic compound is selected from the group consisting ofallyl-, methyl-, ethyl-, bromoethyl-, epoxi-, trimethylsilylethyl-,phenyl-, benzyl-, and a nitrophenyl group.
 5. The method as claimed inclaim 1, wherein said nucleotide sugar is at least one member selectedfrom the group consisting of CMP-Neu5Ac, GDP-Fuc, UDP-Gal, UDP-GlcNAc,and UDP-GalNAc.
 6. The method as claimed in claim 1, wherein at leastone of said glycosidase and at least one of said glycosyltranferase arein soluble or immobilized form and are used simultaneously for thesynthesis of said oligosaccharide compound.
 7. The method as claimed inclaim 1, wherein Neu5Acα2-3Galβ1-3GalNAc-R and Neu5Acα2-3Galβ1-3GlcNAc-Rare synthesized employing β-D-galactosidase and β-D-galactoside(α2-3)sialyl-transferase, wherein R is a glycosidically bound aglycon.8. The method as claimed in claim 1, wherein one ofGalβ1-3(Fucα1-4)GlcNAc-R, Neu5Acα2-6Galβ1-4GlcNAc-R,Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc-R, Fucα1-2Galβ1-3GlcNAc-R,Fucα1-2Galβ1-4GlcNAc-R, Galβ1-4(Fucα1-3)GlcNac-R,Galα1-3Galβ1-4GlcNAc-R, GalNAcα1-3Galβ1-3GlcNAc-R,GalNAcα1-3Galβ1-4GlcNAc-R, ##STR4## is synthesized, wherein R is aglycosidically bound aglycon.
 9. The method as claimed in claim 1,wherein said oligosaccharide compound having bio-specific affinity toanother substance is synthesized and isolated.
 10. A method forenzymatic synthesis of an oligosaccharide compound consistingessentially of reacting a donor substance, wherein said donor substanceis an oligosaccharide or monosaccharide or glycoside and an acceptorsubstance with α- or β-bound aglycon, with an E.C. group 3.2 glycosidaseto form an oligosaccharide compound which is an O--, C--, N--, S-- orF-glycoside of a di- or higher oligosaccharide which is reacted with asugar nucleotide and at least one E.C. group 2.4 glycosyltransferase toform the final, higher oligosaccharide product.
 11. The method accordingto claim 10, which further comprises separating said product from thereaction mixture.